The gene trap approach is based on the integration of a gene trap vector into the genome. This can be done either by electroporation of a plasmid construct or by infection with a viral vector. Commonly used viral gene trap vectors have been shown to select for integrations near the 5' end of genes. To date, no plasmid vector with a similar tendency has been reported. In this paper we describe a new plasmid vector, pKC199beta geo. This vector contained a short splice acceptor fragment from the Hoxc9 gene, a full length lacZ gene, including an ATG, and a reduced activity, mutant neomycin phosphotransferase gene as a selectable marker. This vector enriched the population of trapped genes in our gene trap screen for insertion events in the 5' end of genes. In the two cases examined the beta-galactosidase activity pattern accurately reflected the endogenous promotor activity.